A team representing the Southern Movement, demanding a north-south federation, and another for Zaidi Ansarullah rebels held a sit-in at the Sanaa hall where the meeting was to be held, an AFP reporter said.
This forced organisers to postpone the session until Thursday.
The two groups had boycotted Tuesday's session during which President Abdrabuh Mansur Hadi promised that outstanding issues would be resolved within days.
In a joint statement, the groups said they boycotted the session due to a "lack of consensus" on the northern and southern questions.
They charged that "the most important national issues have been handed over to traditional forces who are dividing its outcomes among themselves."
"It is injustice that has united us," said Mohammed Ali Ahmed, head of the southern delegation and chairman of the People's Congress of the South -- a Southern Movement faction.
The dialogue that opened in March is aimed at drawing up a new constitution for Yemen and preparing for elections in February.
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Fighting between Zaidi Shiites and government forces in the mainly Sunni state after a 2004 rebellion killed thousands of people before a ceasefire was reached in February 2010.
The dialogue was scheduled to end September 18 but delayed after participants, despite having accepted the concept of a federal structure, failed to agree on the numbers of regions making up the future state.
The southern question has been a major stumbling-block in the talks, with hardline factions of the Southern Movement boycotting the discussions and demanding secession.
After the former North and South Yemen united in 1990, the south broke away in 1994, triggering a brief civil war that ended with the region being overrun by northern troops.
"What concerns us is having a single southern entity with the pre-unity borders," said Ahmed, defending the "right of the people to determine their fate."
Hadi on Tuesday said: "We are a few days away from reaching a just solution to the southern issue... within a united and federal Yemen."
Secession of the south would prove "a catastrophe that future generations will never forgive us for."
Separatist groups have called for a rally on Saturday in Aden, capital of the formerly independent south, in support of a complete secession.